1. Technical Field
The present invention relates to a method for producing purine nucleosides, which are important as raw materials in the synthesis of 5′-purine nucleotides, such as 5′-inosinic acid or 5′-guanylic acid, and the present invention further relates to a novel microorganism used for the production.
2. Background Art
Conventionally, purine nucleosides, such as inosine, guanosine, and xanthosine, are industrially produced by fermentation utilizing adenine auxotrophic strains. Alternatively, such strains may be further imparted with drug resistance against various drugs, such as purine analogues and sulfaguanidine. These strains include those which belong to the genus Bacillus (Japanese Patent Publication Nos. 38-23099 (1963), 54-17033 (1979), 55-2956 (1980), and 55-45199 (1980), Japanese Patent Application Laid-Open No. 56-162998 (1981), Japanese Patent Publication Nos. 57-14160 (1982) and 57-41915 (1982), and Japanese Patent Application Laid-Open No. 59-42895 (1984)), or the genus Brevibacterium (Corynebacterium) (Japanese Patent Publication Nos. 51-5075 (1976) and 58-17592 (1972), and Agric. Biol. Chem., 42, 399 (1978)), or the genus Escherichia (WO9903988), and the like.
Typical methods for obtaining such mutant strains include subjecting microorganisms to mutagenesis by UV irradiation or by treating them with nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine) and selecting the mutant strain by using a suitable medium. On the other hand, breeding such mutant strains using genetic engineering techniques has also been practiced for strains belonging to the genus Bacillus (Japanese Patent Application Laid-Open Nos. 58-158197 (1983), 58-175493 (1983), 59-28470 (1984), 60-156388 (1985), 1-27477 (1989), 1-174385 (1989), 3-58787 (1991), 3-164185 (1991), 5-84067 (1993), and 5-192164 (1993)), the genus Brevibacterium (Corynebacterium) (Japanese Patent Application Laid-Open No. 63-248394 (1988)), or the genus Escherichia (WO9903988).
The productivity of purine nucleoside-producing strains could be further improved by increasing the purine nucleoside excretion activity. It is generally accepted that the penetration of metabolites across the cytoplasmic membrane is usually mediated by specific efflux transporter proteins (Pao et al., Microbiol. Mol. Biol. Rev., 62, 1-34, (1998); Paulsen et al., J. Mol. Biol., 277, 573-592 (1998); Saier et al., J. Mol. Microbiol. Biotechnol., 257-279 (1999)). The present inventors have previously shown that inosine- or xanthosine-producing strains belonging to the genus Escherichia or Bacillus which have enhanced activity of the RhtA protein, coded by rhtA (ybiF) gene, produced more inosine or xanthosine than the parental strains (Russian patent No. 2239656). Besides, inosine-producing strains belonging to the genus Escherichia which have enhanced activity of the YijE, YdeD, or YicM proteins, coded by the yijE, ydeD or yicM genes, respectively, produced more inosine than the parental strains (Russian patent Nos. 2244003, 2244004, 2271391, respectively).
The present inventors have previously found that the yeaS (leuE) gene of E. coli encodes a membrane protein which belongs to the RhtB family and is involved in the efflux of amino acids (Aleshin et al., TIBS, 1999). Specifically, this gene encodes an exporter of leucine, histidine, and methionine (Kutukova et al., FEBS Letters, 579, 4629-4534, 2005).